Spectral phasor imaging on a commercial confocal microscope without a spectral detector.

Area of Science:

• Biophysics
• Microscopy
• Spectroscopy

Background:

• Spectral imaging is crucial for analyzing fluorescent probes and identifying species.
• Traditional lambda scan mode in confocal microscopes suffers from poor temporal resolution and photon inefficiency, limiting live sample analysis.
• There is a need for improved spectral imaging techniques compatible with standard confocal microscopes.

Purpose of the Study:

• To develop and validate a novel spectral imaging method using simultaneous 4-channel acquisition and spectral phasor analysis.
• To demonstrate the method's improved performance over lambda scan mode in terms of temporal resolution and photon efficiency.
• To showcase the application of this method for analyzing environment-sensitive probes, spectral unmixing, and multicolor imaging in live biological samples.

Main Methods:

• Implementation of a 4-channel simultaneous image acquisition system on a commercial confocal laser scanning microscope.
• Application of spectral phasor analysis to the acquired 4-channel data for spectral characterization.
• Utilizing environment-sensitive dyes (ACDAN, Nile Red) and fluorescent proteins (mEmerald, EYFP) for method validation.
• Performing spectral unmixing and multicolor imaging in live cells and organoids.

Main Results:

• The 4-channel spectral phasor method offers superior photon efficiency and temporal resolution compared to lambda scan mode.
• The technique can discern spectral emission changes as small as 5 nm between subcellular compartments.
• Successfully decomposed Nile Red signals into two components and enabled 3-color imaging in live organoids.
• Demonstrated effective unmixing of overlapping emission spectra from fluorescent proteins like mEmerald and EYFP.

Conclusions:

• The 4-channel spectral phasor analysis is a viable and efficient spectral imaging technique for standard confocal microscopes.
• This method significantly advances the study of live biological systems by enabling high-resolution spectral analysis and multicolor imaging.
• The technique provides a powerful tool for spectral unmixing and identifying fluorescent species with overlapping spectra.